Inhibition of native TRPC6 channel activity by phosphatidylinositol 4,5-bisphosphate in mesenteric artery myocytes

The present work investigates the effect of phosphatidylinositol-4,5-bisphosphate (PIP₂) on native TRPC6 channel activity in freshly dispersed rabbit mesenteric artery myocytes using patch clamp recording and co-immunoprecipitation methods. Inclusion of 100 μ m diC8-PIP₂ in the patch pipette and bathing solutions, respectively, inhibited angiotensin II (Ang II)-evoked whole-cell cation currents and TRPC6 channel activity by over 90%. In inside-out patches diC8-PIP₂ also inhibited TRPC6 activity induced by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn -glycerol (OAG) with an IC₅₀ of 7.6 μM. Anti-PIP₂ antibodies potentiated Ang II- and OAG-evoked TRPC6 activity by about 2-fold. Depleters of tissue PIP 2 wortmannin and LY294002 stimulated TRPC6 activity, as did the polycation PIP₂ scavenger poly-L-lysine. Wortmannin reduced Ang II-evoked TRPC6 activity by over 75% but increased OAG-induced TRPC6 activity by over 50-fold. Co-immunoprecipitation studies demonstrated association between PIP₂ and TRPC6 proteins in tissue lysates. Pre-treatment with Ang II, OAG and wortmannin reduced TRPC6 association with PIP₂. These results provide for the first time compelling evidence that constitutively produced PIP₂ exerts a powerful inhibitory action on native TRPC6 channels.